Membrane transport proteins, such as transporters, pumps and channels, cannot fully exert their transport activity simply by being translated as polypeptide chains and inserted into membranes, but demonstrate the physiological transport activity only when the proteins are properly folded and localize to membrane domains where the proteins should function. Heterodimeric Amino acid Transporters (HATs) family consists of a light chain, which has a transport function, and a heavy chain, which is essential for the localization to the plasma membrane. HATs play important roles in various physiological processes. The rBAT (heavy chain)-b^0,+AT (light chain) complex, a member of the HAT family, transports cystine and basic and neutral amino acids. Mutations in either b^0,+AT or rBAT lead to cystinuria (renal cystine stones). Since the identification of the rBAT and b^0,+AT complex in the late '90s, hundreds of pathological mutations have been found. The studies of mutations improved the understanding of the biosynthesis and transport mechanisms of the complex. However, the pathological mechanisms of mutations outside the predicted substrate binding sites remain largely unknown. By combining biochemical analysis and cryo-EM structures, we solved the structure and function of rBAT-b^0,+AT and examined their biogenesis. As a result, we found that b^0,+AT recognizes the substrates at different amino acid residues depending on the type of substrate. Furthermore, we identified a Ca²⁺binding site in rBAT, and showed that Ca²⁺-mediated super-dimerization (dimer x dimer) is a key for the formation of higher-order structures of the complex and its localization to the plasma membrane. Accordingly, we elucidated the pathogenesis of HAT by mutations related to biosynthesis, which is different from mutations in the substrate-binding site. This study provides an understanding of the HAT biogenesis and serves as a guide to develop a new therapeutic approach. At molecular basis, the study lights up a novel role of Ca²⁺ on membrane protein biogenesis.